The Enzyme Kinetics of NADH Dehydrogenase After the Addition

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The Enzyme Kinetics of NADH Dehydrogenase After the Addition of the Inhibitory Molecules, EDTA and Mg2+ JI HAE CHUNG, PANAGIOTIS KARAGEORGIOU, PATRICK YANG, NELSON YANG, AND FRANCES LEVITT Department of Microbiology and Immunology, UBC

The oxidation of NADH to NAD+ is done by NADH dehydrogenase, a component of the electron transport chain in Escherichia coli. It has been demonstrated that when E. coli K12 cells were lysed by the lysozyme lysis method, NADH dehydrogenase activity was inhibited. On the contrary, when cells were lysed by French press, NADH dehydrogenase activity was not inhibited. In our investigation, some common components used in cell lysis were examined for their effect on purified NADH dehydrogenase activity. Attempts to partially purify the enzyme to eliminate contaminating factors were unsuccessful and potential causes were examined. It was found that individually, EDTA and MgCl2 both inhibited the activity of NADH dehydrogenase. However, the pattern in the kinetics of inhibition of EDTA and MgCl2 were different and MgCl2 appeared to be a stronger inhibitor of the enzyme at lower concentrations and limiting substrate levels. ______

Previous studies used crude extracts of clarified environment. However, in addition to the stresses supernatants. These extracts were potentially a problem induced on the cells in the French press protocol, high because interference of NADH dehydrogenase by pressures generate heat that may result in inactivated components of the raw lysate could have been and denatured proteins. As well, NADH dehydrogenase significant. As well, although NADH dehydrogenase is an enzyme embedded in the cellular membrane and activity can still be measured from the lysate of cells improper operations of the French press may result in broken by the French press method, this measured large chunks of membranes remaining unbroken, activity may not be optimal and therefore, investigating containing the enzyme, that will end up in the pellet the lysozyme lysis method is critical. We aimed to when centrifuging out the cellular debris. obtain a purer form of NADH dehydrogenase to avoid Inhibition of the interaction of key co-factor ions such interference. This was attempted with the use of with NADH dehydrogenase by the chelating capability the Diethylaminoethyl (DEAE)-cellulose ion exchange of EDTA, accounts for the major caveat of the chromatography column. Ion exchange lysozyme lysis method. Therefore, with the results chromatography is based on the fact that proteins are from our experiment, a future design of a modified ionically charged, and will interact with any material in lysozyme lysis method can be determined such that the solution that has opposite charge. The protein with inhibition of the activity of NADH dehydrogenase is stronger charge affinity will bind longer than ones with minimal. weak affinity and therefore changing the ionic strength MATERIALS AND METHODS of the buffer will elute the bound materials at differing Bacterial Cell Culture and Growth: E. coli K12 B23 was grown in a standard LB medium (10g Tryptone, 5g yeast extract and concentrations. 10g NaCl per liter of water) to an early-late log phase (5 hrs). Using this pure form of the NADH dehydrogenase, Harvest was done by spinning the cells at 5,000 rpm for 10 minutes. we can investigate the kinetics and inhibitory The pellet was resuspended in 25 ml TS50G (50mM Tris Hcl pH 7.9, characteristics of the enzyme by EDTA and Mg2+ and 50 mM Nacl, 10% glycerol) to prepare for cell breakage. Cell Breakage: The resuspended cell pellet was passed through provide their comparison that will lead to future the Y110L Microfluidizer (Duong Lab, Univeristy of British experiments to further optimize the extraction of the Columbia) at 15,000 psi until the suspension became less viscous (~ enzyme from intact cells using the lysozyme lysis 4 passes).The lysate was then centrifuged at 10,000 rpm for 10 method. This method involves using EDTA and MgCl minutes to remove cellular debris, the supernatant was collected for 2 solubization. to aid in the disruption of cell membranes by lysozyme. Membrane Solubilization: The supernatant after cell breakage Gram-negative cells are incubated with lysozyme and was treated with a final concentration of 1% Triton X-100. The are slowly broken by agitating the mixture over a solution was incubated at 4˚C for 1 hr under constant rotation. The period of time until it becomes viscous, indicating cell solution was centrifuged at 48,800 x g for 1 hr to remove any unsolubilized membrane. One hundred µM DTT was added to the breakage and DNA release. In comparison, French supernatant and kept for further purification/enzyme assays. press uses mechanical energy to lyse cells, where DEAE-Cellulose Ion Exchange Purification of Enzyme: Tris bacterial cells are subject to a shift from a high- buffer (10mM Tris, pH 8, 1 litre) and TN10 buffer (10mM Tris, pH pressure environment to a normal atmospheric pressure 8.0 and 1M NaCl) was used to prepare a series of different buffers

FIG. 1 Bradford standard curve with known concentration of BSA.

with different ionic strengths: TN1, TN2, TN4, TN6, TN8. Samples and therefore, we used this technique in order to were loaded in Vivapure minispin DH membrane spin columns (Cat# examine the extent of inactivation of EDTA, Mg2+, and VS-IX01DH24, Vivascience Sartorius Group Inc., Edgewood, NY, USA). All centrifugations were at 5000 x g for 2 minutes. Filtrate polymixin B post lysis. was pooled and saved for Bradford and enzyme assays. The column Upon completion of the Bradford assay, we have was then subsequently washed with increasing concentrations of TN measured the protein concentration of the lysate after buffers. TN samples were saved for Bradford and enzyme assays (8). solubilization and centrifugation to be 12 mg/ml. The Bradford Assay: Protein was measured with the Bradford dye reagent, Coomasie Blue G-250 biding assay with chicken egg high level of proteins within the lysate indicated that albumin as the standard (8). the cell breakage mechanism was sufficient to release NADH dehydrogenase Activity Assay: The assay for NADH the proteins from the cells. dehydrogenase was based on the observation that NADH absorbs Partial purification of NADH dehydrogenase with light at 340nm but NAD does not. This means that a tube containing NADH and NAD dehydrogenase should show a decrease in DEAE cellulose ion exchange column was absorbance which is inversely proportional to the amount of enzyme unsuccessful. In order to avoid loss of NADH converting the NADH to NAD. The spectrophotometer was blanked dehydrogenase recovery from the lysate, the removal of with 1.35 ml of 50 mM Tris-HCl buffer, pH 7.4 and 1.5 ml of dH2O. cellular debris was incubated with Triton X-100. Triton One hundred µl of 5 mg/ml NADH (Sigma) was added to bring the X-100 is a non-ionic detergent that has the ability to absorbance (A340) to 0.6. To each reaction tube, 100 µl of sample was added. The absorbance was continuously monitored in intervals of solubilize membranes, and therefore, release any five minutes using the SpecX software (Vernier Inc, Seattle) (8). membrane coated NADH dehydrogenase in the solution (5). The intent was to partially purify NADH RESULTS/DISCUSSION dehydrogenase in order to perform inhibition assays on a purer form of the enzyme containing higher activity. The aerobic respiratory chain of Escherichia coli is The purification of NADH dehydrogenase by means of composed of a number of membrane-bound, multi- DEAE-cellulose ion exchange chromatography was not subunit enzymes, including nicotinamide adenine successful in our investigation. Sufficient enzyme dinucleotide (NADH) dehydrogenase (type I activity was only observed in the raw cell lysate and dehydrogenase). This enzyme is an oxidoreductase that the flow through of our samples (Fig.2). This means + oxidizes NADH to NAD and reduces ubiquinone-8 to that either our protein was not eluted by our series of ubiquinol-8 within the cytoplasmic membrane (1). TN solutions, or that our protein did not bind to the Previous studies have shown that inactivation of this column at all. A previous study used DTT in the enzyme is caused by the lysozyme lysis method (8) and purification process (3). High concentrations of DTT 2+ more specifically, the EDTA and Mg required in this may have been a problem in previous studies since it method (5). Further, the only lysis method shown to reduces disulfide bonds, causing the protein to lose its conserve enzyme activity is via the French press (5,8) three-dimensional structure. Because of this reason, we left the dithiothreitol (DTT) out of our protocol.

FIG. 2 The observed activity of NADH dehydrogenase after and before treatment on a DEAE ion exchange filter.

However, DTT is commonly used to reduce suppressed roughly 40% of the original activity. disulfide bonds quantitatively and maintain monothiols Further increases of EDTA concentration to 6.3 mM in the reduced state (2). At low concentrations, DTT and 12.6 mM did not have a proportionate increase in stabilizes enzymes and other proteins which possess suppression. There is an optimal Ca2+ requirement for free sulfhydryl groups and has been shown to restore maximal NADPH dehydrogenase enzyme activity in activity lost by oxidation of these groups in vitro. It the plant Helianthus tuberosus, where even minimal seems that without DTT, our NADH dehydrogenase amounts of EDTA will chelate available Ca2+ (7). The may have in fact denatured and lost its activity. We evolutionary conservation of the NADH dehydrogenase should have used a lower concentration of DTT than enzyme suggests that the same may be true for bacterial used in previous studies, instead of leaving it out of the species. It is interesting to note that in the bacteria protocol completely. Also, it is possible that the pH we Neurospora crassa, the mitochondrial NADH used (pH = 7.64) was too low, so our protein was did dehydrogenase shows an internal site that specifically not possess enough negative charge to bind to the binds Ca2+ (6) This fact, in concert with suggested Ca2+ column. Therefore, NADH dehydrogenase activity was regulation of plant NADPH dehydrogenases provides a only observed in the flow through, and in the original strong argument that Ca2+ concentrations will raw cell lysate. However, this is unlikely, since the significantly effect the activity of NADH previous study by Dancey et al. (2) had binding occur dehydrogenase that we isolated from E. coli. At low at a pH of 7.5. It is possible that the material did not substrate levels there seems to be an EDTA elute at the tested salt concentrations, or that the NaCl concentration dependent effect on the enzyme activity. was inhibitory at the level that the elution occurred, When there is 0.6 mM of EDTA with 0.07 mg/ml of causing an absence of enzyme activity in the filtrate. NADH in the assay solution, there is a 67% increase in Spin column membranes tend to bind more strongly enzyme activity compared to the activity of NADH than particulate columns, so it might take higher salt dehydrogenase when incubated with 12.6 mM of concentrations to elute the material out of spin EDTA. However, this difference in inhibition is not as columns.The activity of NADH dehydrogenase does well visualized once we have higher substrate appear to be affected by the addition of DTT (Fig. 3). concentrations (0.16mg/ml, 0.23 mg/ml). This There is a significant decrease in enzyme activity when observation may be caused by substrate saturation at DTT was omitted. Therefore, omitting DTT from our 0.23 mg/ml of NADH. When the enzyme is saturated purification protocol may have resulted in the with substrate, the relative degree of difference of aggregation of available NADH dehydrogenase, inhibition between the different concentrations of leading to a lower measured activity. EDTA added into the assay would not be as great as As figure 4 indicates, EDTA has a significant when there is less substrate in the solution. inhibitory effect on NADH dehydrogenase even at a low concentration of 0.6mM. At 0.6mM, the EDTA has

FIG. 3 The effects of DTT on the enzyme activities of NADH dehydrogenase expressed over various concentrations of NADH substrate.

FIG. 4 The effects of different concentration of EDTA on the enzyme activities of NADH dehydrogenase expressed over various concentrations of NADH substrate. EDTA significantly inhibits the activity of NADH dehydrogenase by chelating available Ca2+ ions.

When the concentration of EDTA increases, we observation is valid because the inhibitory have an increased Km (Fig. 5). Furthermore, as the characteristics of EDTA would result in a higher EDTA concentration increases, there is a decrease in amount of substrate needed for the enzyme to reach its the Vmax (the maximum enzyme activity) of the Km. Consequently; the maximum activity of the enzyme. The Km indicates the amount of substrate enzyme is also reduced. required for ½ of the maximal enzyme activity. This

FIG. 5 Lineweaver-Burk plots of 1/enzyme activity and 1/[S] to determine the theoretical maximum rate (VMax) and Km values for the enzyme activity of NADH dehydrogenase teated with different concentrations of EDTA.

FIG. 6 The effects of different concentration of Mg2+ on the enzyme activities of NADH dehydrogenase expressed over various concentrations of NADH substrate.

2+ Mg addition to NADH dehydrogenase resulted in MgCl2 (0.7 mM, 6.7 mM, and 13 mM) that were an similar inhibition to that by EDTA. There appears implemented in the experimental protocol. Even at the to be little difference in the inhibition of NADH lowest concentration, the presence of Mg2+ ions dehydrogenase by the three different concentrations of decreased the observed activity of NADH dehydrogenase by 40%. The lack of significant

FIG. 7 Lineweaver-Burk plots of 1/enzyme activity and 1/[S] to determine the theoretical maximum rate (VMax) and Km values for the enzyme activity of NADH dehydrogenase teated with different concentrations of Mg2+.

differences in inhibitory activities between the different pattern indicates that Mg2+ is a stronger inhibitor than concentrations of MgCl2+ used and at different EDTA at lower concentrations. While both of the substrate concentrations could mean that we were inhibitors will decrease the activity of NADH already at saturation of both the inhibitor and substrate dehydrogenase significantly (both suppresses enzyme levels (NADH substrate quantities used in the Mg2+ activity to about 40% of the control, uninhibited experiments were the same as the experiments with enzyme), at limiting substrate concentrations, Mg2+ will EDTA). Therefore, any further increase in substrate or inhibit the enzyme more efficiently than EDTA. The inhibitor would not have made much difference in our inhibitor constants, KI, for EDTA and Mg2+ were of data plot. values 3.1 and 3.5. The similarity in KI values indicate It is seen in literature that Mg2+ can act as an that The slightly higher KI value for Mg2+ suggests that inhibitor to certain enzymes that are analogous to Mg2+ is a stronger inhibitory reagent for NADH NADH dehydrogenase. Mg2+ has been postulated as a dehydrogenase. ligand of glutamate dehydrogenase that regulates its activity. However, the level of inhibitory effect of Mg2+ FUTURE EXPERIMENTS on glutamate dehydrogenase is regulated by ADP/ATP ratios. ADP is an allosteric activator of glutamate In the future, since we postulated that Ca2+ may be a dehydrogenase. In the absence of ADP, Mg2+ is an component required for the activity of NADH inhibitor of the enzyme. (4). Therefore, by a similar dehydrogenase, additional experiments should assess mechanism, Mg2+ can potentially inactivate NADH whether it is the Mg2+ competing with, or the EDTA dehydrogenase at the concentrations that we used. chelating the available Ca2+ that results in a lowered Since Mg2+ had similar inhibition patterns as activity. Another parameter that should be assessed is EDTA, the Lineweaver-Burk plot for Mg2+ resembles whether the Cu2+ binding site found in E. coli NADH that for EDTA; when the concentration of Mg2+ dehydrogenase II (9) contributes to the activity of the increased, the Vmax of the enzyme decreased while the enzyme. An interesting question to ask is whether it is Km increased. These results were expected of the chelation of Cu2+ or Ca2+ by EDTA that is inhibitory compounds. responsible for the decreased enzyme activity measured For EDTA, the Km values were almost identical for in lysates with the addition of EDTA. These the control (no inhibitor), 0.7 mM and 6.7 mM but 13 experiments could be done by the addition of the mM showed a higher Km value. For Mg2+, 0.7 mM and individual components in different combinations into a the control had similar Km values while 6.7 and 13 purified extract of E. coli lysate and measuring the mM had similar, but higher values. This difference in activity of NADH dehydrogenase.

Furthermore, experiments could be done where we REFERENCES try to determine if there are any co-factors that could 1. Calhoun, M.W., G. Newton, and R.B. Gennis. 1991. Physical activate NADH dehydrogenase. It has been discovered Map Locations of Genes Encoding Components of the Aerobic that for glutamate dehydrogenase, ADP could serve as Respiratory Chain of Escherichia coli. J. Bacteriol. 173:1569- a co-factor that would in turn transform the Mg2+ that 1570. was previously inhibitory to the enzyme to an enzyme 2. Cleland, W.W. 1964. Dithiothreitol, A New Protective Reagent for SH Groups. Biochem. 3: 480-2. activator, increasing glutamate dehydrogenase’s 3. Dancey, G., Levine, A., and Shapiro, B. 1976. The NADH activity (5). Since we have postulated that the mode of dehydrogenase of the respiratory chain of Escherichia coli. J. action by Mg2+ on NADH dehydrogenase could be Biol. Chem. 251: 5911-5920. analogous to the divalent cation’s inhibitory effect on 4. Fahien L.A., Teller J.K., Macdonald M.J., and Fahien C.M. 1990. Regulation of glutamate dehydrogenase by Mg2+ and glutamate dehydrogenase, a co-factor that can activate magnification of leucine activation by Mg2+. Mol. Pharmacol. glutamate dehydrogenase could potentially increase 37: 943-9. NADH dehydrogenase’s activity. 5. Madden, M., Song, C., Tse, K., and Wong, A. 2004. The 2+ Inhibitory Effect of EDTA and Mg on the Activity of NADH Dehydrogenase in Lysozyme Lysis. J. Exp. Microb. Immun. ACKNOWLEDGEMENTS 5:8-15. 6. Melo, Ana M.P. et al. 1999. Primary structure and We would like to thank the following individuals: characterisation of a 64 kDa NADH dehydrogenase from the Franck Duong in the Department of Biochemistry for inner membrane of Neurospora crassa mitochondria. Biochimica et Biophysica Acta 1412: 282-287. kindly lending us the Microfluidizer Y110L for our cell 7. Møller, I. M., Johnston, S. P., and Palmer, J. M. 1981. A breakage protocol. Karen Smith, Gaye Sweet and specific role for Ca2+ in the oxidation of exogenous NADH by Jennifer Sibley for providing invaluable advice, Jerusalem-artichoke (Helianthus tuberosus) mitochondria. J. knowledge, assistance and guidance in our Biol. Chem. 194:487–495. 8. Ramey, W.D. 2002. Experiment C2: Effect of the method of experimentations. Lando, Gilly, Loida, Ed, Nick, and cell breakage on the distribution patterns of enzyme activity all other staff in the media room for assisting us with observed after ultracentrifugation, p. 1-10. In Microbiology the materials that we need. Last but not least, we would 421: Source experiments for experimental projects. Department like to thank Dr. William Ramey for his patience, of Microbiology and Immunology, the University of British Columbia, Canada. wisdom, guidance and inspiration that made this project 9. Rapisarda, Vivianna A., et al. 2002. Evidence for Cu(I)-thiolate possible. ligation and prediction of a putative copper-binding site in the Escherichia coli NADH dehydrogenase-2. Arch. Biochem. Biophys 405: 87-94.